8.4.2 YAP介导ATO调节VSMC的表型转化
体外利用不同浓度的ATO（0、2、4和6 μmol/L）处理A7r5大鼠平滑肌细胞24 h后，进行YAP免疫荧光染色。图8-7（a）的结果表明：随着药物浓度的增加，YAP总量以及进入细胞核的YAP量逐渐减少。同样，体内实验（支架植入1周）进一步证实：ATO（AES组：ATO药物洗脱支架）在抑制VSMC增生时减少了YAP的入核反应（图8-7（b））。WB分析结果可知（图8-7（c）），ATO抑制YAP表达时促进了收缩表型因子SM22α的表达，其合成表型因子骨桥蛋白（osteopontin, OPN）的表达随之降低。结果表明：ATO在调节细胞表型向收缩型转化的过程中，YAP进入细胞核的量以及蛋白表达量有所降低。从而暗示：Hippo/YAP信号通路很可能响应了细胞的机械力学改变并介导ATO诱导VSMCs由合成型向收缩型的转化。
图 8-7 YAP参与ATO诱导VSMC表型调节。（a）ATO处理A7r5 1 d后YAP免疫荧光染色，标尺为20 μm。（b）颈动脉支架植入1周后横断面行YAP免疫荧光染色。（c）WB检测ATO作用1 d后YAP、SM22α和骨桥蛋白（OPN）的表达水平。A0, A2, A4和A6代表ATO的浓度分别为0、2、4和6 μmol/L；BMS（裸金属支架）、PMS（聚合物涂层金属支架）、AES（三氧化二砷药物洗脱支架）；p值＜0.05（*），＜0.01（**），＜0.001（***）。［引自：Zhao Y, et al. A novel mechanism of inhibiting in-stent restenosis with arsenic trioxide drug-eluting stent: enhancing contractile phenotype of vascular smooth muscle cells via YAP pathway［J］. Bioact Mater, 2021, 6（2）: 375-385.］
Figure 8-7 YAP is involved in ATO-induced VSMC phenotype modulation.（a）Immunostaining of YAP after ATO treating A7r5 for 1 d, scar bar =20 μm.（b）Immunostaining for YAP in cross-sections of carotid arteries post-implanting stent for 1 week.（c）Protein expression levels of YAP, SM22α and Osteopontin（OPN）were determined by WB after ATO treating for 1 d. A0, A2, A4 and A6 represent 0, 2, 4 and 6 μmol/L of ATO respectively; BMS（bare metal stent）, PMS（polymer coating-metal stent）, and AES（arsenic trioxide-drug eluting stent）; p values＜0.05（*）,＜0.01（**）and＜0.001（***）. ［Adapted from: Zhao Y, et al. A novel mechanism of inhibiting in-stent restenosis with arsenic trioxide drug-eluting stent: enhancing contractile phenotype of vascular smooth muscle cells via YAP pathway［J］. Bioact Mater, 2021, 6（2）: 375-385.］(https://www.daowen.com)
有趣的是，由图8-8可以看出磷酸化的YAPp-YAP与细胞骨架F-actin有共定位现象，两者在细胞内的分布有很大的相关性，且细胞骨架聚合程度与p-YAP的分布与排列一致。总之，ATO处理A7r5平滑肌细胞随药物浓度的增加、p-YAP的表达增强，且p-YAP的分布与细胞骨架排列密切相关。进一步利用WB实验验证p-YAP与ROCK和细胞表型相关因子的相关性，结果表明YAP介导VSMCs表型转化是通过或部分受到ROCK通路的调节。
图8-8 体外YAP活性（p-YAP）介导ATO调节VSMCs的表型转化。（a）ATO作用于A7r5 24 h 后F-actin与p-YAP免疫荧光共染色，标尺长度均为20 μm。（b）F-actin和p-YAP免疫荧光强度统计图。（c）为WB实验验证ROCK抑制剂处理后对YAP活性及VSMCs表型因子的调节作用。（d）为WB实验炎症YAP抑制剂处理后YAP活性及VSMCs表型因子的调节作用。（e）YAP和ROCK在ATO调节VSMCs分化中的作用。其中，A0、A2、A4和A6代表ATO的浓度分别为 0、2、4和6 μmol/L。p值＜0.05（*）、＜0.01（**）、＜0.001（***）、＜0.000 1（****）。［引自：Zhao Y, et al. A novel mechanism of inhibiting in-stent restenosis with arsenic trioxide drug-eluting stent: enhancing contractile phenotype of vascular smooth muscle cells via YAP pathway［J］. Bioact Mater, 2021, 6（2）: 375-385.］
Figure 8-8 The activity of YAP is associated with ATO regulating the phenotype transformation of VSMCs in vitro.（a）Immunofluorescence co-staining of F-actin and p-YAP after ATO treating A7r5 for 1 d, scar bar =20 μm.（b）The fluorescence intensity of F-actin and p-YAP.（c）VSMCs（A7r5 cell lines）were treated with control or ATO for 1 d with or without ROCK inhibitor Y27632 and WB experiments for indicated the protein levels of p-YAP and SM22α were performed.（d）VSMCs（A7r5 cell lines）were treated with control or ATO for 1 d with or without YAP inactivator verteporfin and WB experiments for indicated the protein levels of p-YAP and SM22α were performed.（e）Working hypothesis which elaborates role of YAP and ROCK in ATO modulation the differentiation of VSMC. A0, A2, A4 and A6 represent 0, 2, 4 and 6 μmol/L of ATO respectively; “ns” means no significance, p values＜0.05（*）,＜0.01（**）,＜0.001（***）and＜0.000 1（****）. ［Adapted from: Zhao Y, et al. A novel mechanism of inhibiting in-stent restenosis with arsenic trioxide drug-eluting stent: enhancing contractile phenotype of vascular smooth muscle cells via YAP pathway［J］. Bioact Mater, 2021, 6（2）: 375-385.］